Production of materials having an anisotropic structure

ABSTRACT

The present invention relates to a process for the production of a multi-layered material having anisotropic pores. It further relates to a multi-layered material which can be produced by the process according to the invention, and to the use of a multi-layered material as a chondral support matrix, a meniscus support matrix or an intervertebral disc support matrix.

RELATED APPLICATIONS

This application is a 371 National Stage Application of InternationalApplication Number PCT/EP2012/074980, filed 10 Dec. 2012, which claimspriority to European Application Number 10 2011 120 488.5 that was filedon 8 Dec. 2011, the contents of which are incorporated by referenceherein.

FIELD OF THE INVENTION

The invention relates to the field of production of materials, inparticular the production of medical materials.

BACKGROUND OF THE INVENTION

Medical materials are used, for example, for implants, microsensors andother products which are placed in the human or animal body. In thiscontext the medical materials directly come into contact with the tissueand the cells of the body. In their natural environment cells aresurrounded by an extracellular matrix which is important for thesurvival of the cells, since it decisively influences their adhesion,proliferation, migration, differentiation and function. The maincomponents of the extracellular matrix are hydrogels and polymer fibreswhich are not water-soluble, these serving as a mechanical scaffold.Basal membranes and ultrathin separating layers between tissues are alsopresent. These structures are matched to the different biologicalrequirements of various organs and tissues. In order to promote a goodinteraction between the cells and the medical materials, attempts aremade to adapt the structure and composition of the materials to thenatural environment of the cells.

DE 19751031 A1 describes highly pure collagen sponge products having anopen pore structure which shall enable growth of the cells into thesponge. A freezing process with which a homogeneous and targeteddistribution of the collagen fibres in channel-like guiding structuresis generated by finger-shaped ice crystals growing through a collagentype I dispersion is used for production of the collagen sponges. Forthis, analogously to the common processes of metalworking, freezingprocesses have been designed which structure a mixture of substancesbetween two temperature-controllable surfaces which are arrangedparallel or concentrically with respect to one another by keeping thetemperature gradient between the surfaces constant. The mixturestructured in this manner is then freeze-dried by precooling it underoverpressure and releasing the pressure suddenly. However, the materialsobtained in this way exclusively consist of one single functionalcomponent, namely collagen type I, and therefore can reproduce thenatural environment of cells to only a limited extent.

Tampieri et al., 2008 describe the combination of three layers ofcollagen scaffolds of different composition to form an overallstructure, each scaffold representing a different bone or cartilagelayer. The top layer consists of pure collagen I and serves as achondral zone replacement. Collagen I mineralized by a precipitationreaction with hydroxylapatite, with a mineral-matrix ratio of 70/30 wt.%, replaces the subchondral bone. However, an intermediate layermineralized in the same manner, in a mineral-matrix ratio of 40/60 wt.%, differs significantly from the tidemark of native chondral tissue.The hydroxylapatite of the subchondral zone here is partially doped bymagnesium. The individual collagen scaffolds were each crosslinkedseparately by 1,4-butanediol diglycidyl ether (BDDGE) and bonded to oneanother using a “weaving process”. The overall structure was thenfreeze-dried. Cell experiments showed, however, that the structureproduced in this way allows only a limited population by cells. Thus,only a low cell migration into the inside of the chondral replacementzone with a non-uniform distribution was to be observed, whereas theregion in the centre of the scaffold remained substantially acellular. Acomplete integration of the material after implantation is therefore notpossible.

EP 1858562 B1 describes a porous, three-layered, osteochondral scaffold.This comprises a zone covered with a smooth surface, which is made tothe extent of 100% of collagen I of equine origin and shall correspondto chondral tissue. The region of the subchondral and osteal zone isrepresented by composites of collagen type I and nanostructured,magnesium-enriched hydroxylapatites. The individually produced zones,however, are joined together only subsequently by a compressionoperation, as a result of which a delamination may occur duringrehydration of the freeze-dried matrices. Moreover, histological resultsof animal studies show that only fibrous chondral tissue but no nativearticular chondral tissue is formed in the chondral zone. Such fibrouschondral tissue is formed above all during the natural but rare andincomplete self-healing processes of the cartilage. In addition to thecollagen type II mainly occurring in healthy articular cartilage, italso comprises atypical collagen type I and differs decisively instructure and functionality from native chondral tissue. The adverseformation of fibrous chondral tissue is possibly to be attributed to thefact that the chondral replacement zone only consists of one individuallayer, which has an atypical composition compared with native cartilageand too large porosities with an unnatural alignment.

There is therefore the need for stable materials which have functionallydifferent regions which reproduce the natural environment of cells.

SUMMARY OF THE INVENTION

The invention relates to a process for the production of a multi-layeredmaterial having anisotropic pores, comprising the steps of providing atemperature gradient by two temperature-controllable bodies arrangedopposite one another; arranging and solidifying in the temperaturegradient a first substance which contains at least one sublimablecompound in order to form a first layer; arranging and solidifying inthe temperature gradient at least one further substance which containsat least one sublimable compound in order to form at least one furtherlayer adjacent to the preceding layer; subliming the compound andconsolidating the layers.

The invention also relates to a multi-layered material which can beproduced by the process according to the invention, and to a medicalmaterial having at least two different layers, in which pores extendanisotropically through at least two layers of the material. Theinvention furthermore relates to the use of a multi-layered material asa chondral support matrix, a meniscus support matrix or anintervertebral disc support matrix in which pores extend anisotropicallythrough at least two layers of the material.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure and the fundamental mode of functioning of apossible embodiment of a solidification apparatus. a) Simplifiedschematic representation of the mode of functioning. Peltier elementscoupled to heat exchangers ensure, via a controlled external temperaturegradient, an anisotropic crystal growth in the substances in the samplechamber. b) Assembling plan of a possible embodiment of a solidificationapparatus. The sample chamber is in the centre of the insulation unit I1and is in thermal contact with the temperature-controllable bodies W1and W2, which are located above and below the sample chamber. Thetemperature-controllable bodies are within the insulation unit I2 andare coupled to the Peltier elements P1 and P2, which are fixed by theinsulation units I3 and I4. This inner assembly is in the centre of theheat exchanger ring A2, which together with the heat exchanger units A1and A3 forms an outer assembly.

FIG. 2 shows a histological (left) and a schematic (middle)representation of the native articular cartilage structure with threedistinguishable zones (s: superficial; m: middle; d: deep) (Klein etal., 2009), and a biomimetic support matrix built up in layers andhaving continuous pores (right). Broken lines running vertically incurves in the schematic representation of the articular cartilagestructure symbolize the orientation of collagen fibres. The chondrocytesare adapted differently in their shape (spheroids) and organization tothe specific requirements of the particular zone (s, m and d). In thesupport matrix the bone substitute composite (SC) is joined to a deeperchondral zone (CD), which passes into a middle chondral zone (CM). Thisis in turn joined to functionalized polymer fibres, which form a finalsliding layer (CS).

FIG. 3 shows light microscopy (a), a photographic (b) and an electronmicroscopy image (c) of a monolithic, osteochondral alginate-basedsupport structure. Anisotropic pores having a cross-section in theregion of 80 μm in size run continuously through the individual zones ofthe chondral part (CM—upper zone shown as dark and CD—middle zone shownin grey) and through the subchondral part (SC—lower zone shown inwhite). The subchondral part is mineralized by the absorbable calciumphosphate phase bruschite. Individual zones were produced with colouredprecursors.

FIG. 4 a shows an electron microscopy image of the surface of a chondralsupport matrix produced according to Example 2. FIG. 4 b shows ahistological section, stained with haematoxylin-eosin, through thecentre of the chondral zone of an alginate matrix populated for 21 daysby human mesenchymal stem cells. After static cell population, the cellshave migrated into the inside of the support matrix and have adheredthere preferentially to microstructures within the pores. Synthesisproducts in the form of matrix formed by cells are already to be seen.FIG. 4 c shows a light microscopy image of a plan view of acollagen-based chondral support structure. The support matrixpredominantly consists of collagen and chondroitin sulphate and istraversed by anisotropic, longitudinal pores. FIG. 4 d shows a scanningelectron microscopy image of a cross-section through the support matrixof FIG. 4 c.

FIG. 5 shows a schematic representation of a support matrix fortreatment of meniscus defects (a), the geometry of atemperature-controllable body (bottom) and an insulating body (top) forthe production of such a support matrix (b) and an alginate model of alateral meniscus (c). The line drawn in the part image c illustrates thecourse of the pore structure. The support matrix consists of an outermeniscus region (OM) and an inner meniscus region (IM), which differ intheir chemical compositions.

FIG. 6 shows scanning electron microscopy images of a vertical sectionthrough a collagen support matrix for treatment of meniscus defects(Example 4).

FIG. 7 a shows the schematic representation of a support matrix fortreatment of intervertebral disc defects. Analogously to native tissue,the support matrix has constituents of the extracellular cartilagematrix, which are combined into a biomimetic, monolithic matrix. Anon-aligned network which corresponds to the nucleus pulposus (NP) is tobe found within a fibre arrangement of lamellar structure produced byaligned solidification, which corresponds to the annulus fibrosus. Thisis in turn divided into an outer annulus fibrosus (oAF) and an innerannulus fibrosus (iAF). The detailed view shows the main structure ofthe microstructure of the annulus fibrosus, which consists of individuallamellae joined to one another. FIGS. 7 b and c show a monolithic,intervertebral disc-like support structure based on alginate,anisotropic lamellae characterizing the structure of the outer region ofthe support matrix.

FIG. 8 shows a scanning electron microscopy sectional image offunctionalized polymer fibres which form the final sliding layer (CS) ofan underlying osteochondral support matrix and are joined to the middlechondral zone (CM). The polymer fibres run orthogonally to the porestructure of the remaining support matrix and in this way reproduce thefibre arrangement of native osteochondral tissue.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the invention relates to a process for the productionof a multi-layered material having anisotropic pores, comprising thesteps of providing a temperature gradient by twotemperature-controllable bodies arranged opposite one another; arrangingand solidifying in the temperature gradient a first substance whichcontains at least one sublimable compound in order to form a firstlayer; arranging and solidifying in the temperature gradient at leastone further substance which contains at least one sublimable compound inorder to form at least one further layer adjacent to the precedinglayer; subliming the compound and consolidating the layers.

The process serves for production of a material which comprises several,that is to say at least two, preferably three, different layers whichare combined to a monolith. The layers can differ in their composition,their functionality and their physical properties. Inasmuch they aresimilar to biological tissues, such as, for example, extracellularmatrices, which likewise have regions or layers of different chemicalcomposition and biological functionality. The materials produced by theprocess are accordingly suitable in particular for medical applications,also within the animal or human body. Due to the different layers theycan reproduce the natural environment into which they are introduced,and where appropriate perform corresponding biological functions.

The different layers are formed by arranging several substances over oneanother or side by side and solidifying them. In order to obtain acontinuous anisotropic pore structure, the substances are arranged onone another or side by side without the solidification process beinginterrupted.

The substances are solidified by being arranged in a temperaturegradient formed by two temperature-controllable bodies arranged oppositeone another. A solidification process here is to be understood asmeaning that the degrees of freedom for spatial movement of particles isrestricted such that these can still move spatially to only a very smallextent, if at all. In many cases this is accompanied by a phaseconversion. The solidification can be carried out in an aligned ornon-aligned manner, a macroscopically isotropic structure being formedin a non-aligned solidification and a macroscopically anisotropicstructure being formed in an aligned solidification. By the substancesbeing solidified in a directional temperature gradient, an alignedsolidification occurs, the solidification starting at the points of thesubstances with the lowest temperature and moving with time in thedirection of the points of the substances with the highest temperature.The solidification front thereby migrates uniformly through thesubstances. The process can be carried out, for example, with asolidification apparatus as shown in FIG. 1.

During solidifying, the sublimable compound in the substances formscrystals, which can likewise grow in an aligned manner along thetemperature gradient. By the layers being arranged in succession, thecrystals of the sublimable compound grow uniformly successively throughall the layers (FIG. 1 a). Due to the sublimation, the crystals of thesublimable compound are removed from the layers formed, so that hollowpores remain. Because of the aligned solidification, the pores have ananisotropic structure and run continuously through all the layers. Acontinuous anisotropic pore structure enables the population of thematerial by cells as far as the inside, as a result of which a goodintegration and functionality of the material in the native tissue isenabled.

The solidified layers and polymer fibre layers applied can beconsolidated by suitable processes by forming additional linkagesbetween the polymers or between the structures formed by them. Due tothese additional bonds a three-dimensional network is formed, whichincreases the hardness and stability of the material. The consolidatingcan be carried out by various chemical processes, for example by wetchemical crosslinking, dehydrothermal processes, enzymatic crosslinking,non-enzymatic glycation, UV irradiation, gamma irradiation, sintering,infiltration of the material or by a combination of various processes.The wet chemical crosslinking is preferably carried out by means ofcarbodiimides, isocyanates, complexing ions or glutaraldehyde, andfurther preferred under a pressure of ≦300 mbar. In a preferredembodiment consolidating the layers comprises a wet chemicalcrosslinking and a dehydrothermal process.

In a preferred embodiment the temperature gradient is between 0.5 K/mmand 200 K/mm, preferably between 2.5 K/mm and 25 K/mm, further preferredbetween 5 K/mm and 15 K/mm. Such gradients allow a continuous alignedcrystal growth which leads to the formation of anisotropic pores. Thetemperature gradient is determined by the temperatures of the twotemperature-controllable bodies and their distance from one another, onetemperature-controllable body having the lowest temperature and theopposite one the highest temperature and thus generating the gradient.The temperature gradient decisively determines the rate at which thesubstances solidify and therefore also influences the formation of thecrystals of the sublimable compound. Since the crystals of thesublimable compound directly determine the form and size of the pores,the shape of the pores can be influenced by the temperature gradient.Thus, for example, greater temperature gradients lead to the formationof smaller and narrower pores during solidifying of the same substance.Greater gradients therefore tend to be employed at lower temperatures.At lower temperatures, furthermore, greater temperature gradients mayform if the warmer temperature level has an upper limit because of thepossibility of protein denaturings. A small temperature gradient maylead to a slow rate of freezing, which promotes the formation ofcrystals having a columnar morphology. A rapid rate of freezing, on theother hand, may lead to the formation of dendritic crystals with manybranchings. This can be utilized to reproduce a natural tissue structurewhich comprises both isotropically and anisotropically structuredregions, such as, for example, an intervertebral disc. An isotropic porestructure can be generated by equiaxial dendritic crystal growth, whilean anisotropic pore structure can be generated by columnar or dendriticcrystal growth. Materials having anisotropic pores have a higherstability under the particular loads such as occur in specializedtissues compared with those having a non-aligned, isotropic porestructure, and have a higher compressive and tensile strength at thesame porosity. Furthermore, anisotropic pores, in contrast to isotropicpores, allow an effective cell migration into the inside of thematerial. If the material is nevertheless to have isotropic pores, forexample in order to reproduce a natural isotropic structure, this can beachieved by a non-aligned solidification of the substances. For this,the substances are solidified at a uniform temperature or with a verysmall temperature gradient of <0.5 K/mm. It is also possible to formregions or layers having an isotropic structure and those having ananisotropic structure within the same material by subjecting individualregions of the material to aligned or non-aligned solidification.

In a preferred embodiment the substances are solidified with a linearlyinterpolated cooling rate of from 2 K/min to 45 K/min, more preferred 5K/min to 35 K/min. The cooling rate is to be understood as meaning thelinear interpolation of the temperature difference per unit time of thesubstances to be solidified, from the start of the crystal growth tocomplete solidification.

In an advantageous embodiment the temperature gradient is constantduring the process. This means that the temperature difference betweenthe lowest temperature, that is to say the one temperature-controllablebody, and the highest temperature, that is to say thetemperature-controllable body arranged opposite, and the spatialdistance between the two remain unchanged. A stable crystal growththrough all the layers is thereby ensured. A constant temperaturegradient also includes the lowest and the highest temperature, at aconstant distance between the two temperature-controllable bodies, beingincreased or lowered in parallel.

In an advantageous embodiment of the process the temperature gradient,at a constant distance between the temperature-controllable bodies, isincreased or reduced during the process by increasing or lowering thetemperature of the temperature-controllable bodies. Preferably, this iscarried out after the solidification of one or more layers and beforefeeding in a further substance.

In a preferred embodiment the lowest temperature of the temperaturegradient is between −200° C. and +90° C. and the highest temperature ofthe temperature gradient is between +100° C. and −25° C. In a furtherpreferred embodiment the lowest temperature of the temperature gradientis between −60° C. and −15° C. and the highest temperature of thetemperature gradient is between +30° C. and +5° C. The lowest and thehighest temperature of the gradient depend on the composition of thesubstances and the melting point of the sublimable compound.Compositions of high density, for example compositions having a highcontent of polymers, in particular solidify slowly and are thereforesolidified under temperature gradients which are generated at lowertemperatures. If the substances comprise a sublimable compound of lowmelting point, for example an organic solvent, the substances arepreferably solidified at correspondingly low temperatures. If, on theother hand, sublimable compounds such as, for example, water or aceticacid, are used, higher temperatures are possible. Due to theconstitutive supercooling at the solidification boundary, crystalformation of water at above 0° C. is also possible.

In a preferred embodiment the lowest and the highest temperature of thetemperature gradient are constant during the process. The temperaturegradient, that is to say the temperature of the twotemperature-controllable bodies and their distance from one another, isset before arranging the first substance. During the solidification ofthe substances both the lowest and the highest temperature of thetemperature gradient, that is to say the temperatures of thetemperature-controllable bodies, and the distance of the bodies from oneanother remain unchanged. A lowering of the temperatures of the twotemperature-controllable bodies at a constant temperature differenceduring the freezing operation is possible but not necessary.

The term “temperature-controllable bodies” describes both bodies whichcan actively remove heat, such as, for example, Peltier elements, andthose bodies which indirectly remove heat by being cooled or supply heatby being heated. The temperature-controllable bodies are preferably madeof metal or metal compounds. The bodies can be arranged bothhorizontally and vertically, in a horizontal arrangement the lowertemperature-controllable body as a rule determining the lowesttemperature of the temperature gradient. The temperature-controllablebodies can be present in individual geometries, for example as anegative form for the material to be produced. In addition toconventional forms, such as cuboids, cylinders, pyramids, cones,rotational ellipsoids, spheres, rings or subsets thereof in solid formor hollow body form, individualized forms are also possible. The lattercan be produced, for example, according to information fromthree-dimensional imaging methods, such as x-ray tomography or magneticresonance tomography, so that materials adapted individually for eachpatient can be produced. In addition to temperature-controllable bodies,insulating bodies can also be used for shaping the material. These aresuitable above all for giving individual layers or the entire materialan individual form. The temperature-controllable or insulating bodiescan be in direct thermal contact with the substances.

In a preferred embodiment the substances are arranged in a containerwhich is placed in the temperature gradient. In this case the materialis formed within the container and its form is determined by thegeometry thereof. Like the temperature-controllable and insulatingbodies, the container can also serves as a negative form for thematerial and accordingly have the abovementioned forms.

In a preferred embodiment the first and/or further substance,independently of each other, is a solution, a dispersion, a suspension,a gel, a polymer melt, or a mixture thereof. The term “substance”describes the flowable precursors of the layers of the later material.

In a preferred embodiment the first and/or a further substance,independently of each other, contains at least one polymer or monomersthereof. The composition of the precursors determines the composition ofthe individual layers of the material. The constituents of thesubstances are therefore chosen according to the function and theproperties which the individual layers are to have in the latermaterial. Preferably, the layers correspond to the tissue which thematerial replaces or into which it is integrated. Polymers are preferredconstituents of the substances, since they form stable structures andnetworks by intermolecular bonds. Preferably, the polymer is a nativepolymer. In contrast to denatured polymers, native polymers are presentin their natural secondary structure, which enables the molecule theeffective formation of complexes and networks, and can be recognized bycells as native environment. Furthermore, prepolymers and macromonomerscan be used, the prepolymer being a synthetic (co)polymer having amolecular weight of less than 50 kDa, which in addition containscrosslinkable groups, such as, for example, (meth)acrylates, thiols,isocyanates, azides, ethynes, aldehydes, carboxylic acids and/or amines.

In a preferred embodiment the polymer is selected from the groupconsisting of peptides, proteins, preferably structural proteins, andpolysaccharides. The polymer can be a polymer produced by synthesis. Theuse of proteinogenic polymers, in particular the use of structuralproteins, is preferred above all for medical application, since thesepolymers are also present in the natural tissue matrix. By using suchpolymers for the production of the material it is possible to reproducethe body's own structures, also with respect to their chemicalcomposition. Furthermore, proteinogenic polymers, in particularstructural proteins, form stable networks, which are particularlysuitable for the production of materials. Collagens form triple helices,which combine to form long fibres, while keratins form superhelices,which in turn form intermediate filaments. In addition to proteinogenicpolymers, polysaccharides are also suitable for the formation of stablematerials, since they likewise form intermolecular networks, such asmicrofibrils. The formation of such structures contributes decisively tothe stability of the material. In addition to structure-formingpolymers, the substances can also contain further constituents, forexample of the extracellular matrix. The use of glycoaminoglycans, suchas, for example, hyaluronic acid and chondroitin sulphate, which due totheir high degree of hydration can store large amounts of water whichexceed several times their own volume, is particularly preferred. Sincethe natural tissue matrix is highly water-retentive, it is advantageousto incorporate such compounds into the material. By using such polymers,furthermore, an electrostatic repulsion which occurs increasingly duringdeformation in an aqueous medium can be utilized, which can generate ashock-absorbing effect of the support matrix. The physical properties ofthe material are thereby adjusted to the natural tissue matrix.

In a preferred embodiment the polymer is selected from the groupconsisting of collagen type I, II, III, V, VI, IX, X, XI, XII, XIV, XVI,chondroitin sulphate, aggrecan, keratan sulphate, hyaluronic acid,proteoglycan 4, cartilage oligomeric matrix protein (COMP),fibromodulin, procollagen II, decorin, anchorin, hyaluronate, biglycan,thrombospondin, fibronectin, chondrocalcin, alginate, cellulose andchitosan, polylactic acid (D and/or L), polyglycollic acid, copolymersthereof, polycaprolactone, polyanhydrides, polyacetals and polyketals,polyethylene glycol, poly(meth)acrylates, poly(glycidol), aromaticpolyesters, PET, polyoxacyclines, polyurethanes, polyvinyls, polyvinylalcohols, cartilage fragments, collagen fibrils and mixtures thereof.The composition of the substances can be chosen according to thebiochemical composition of the tissue which the material is toreproduce, for example different cartilage tissue or bone. For theproduction of model matrices, the polymer is preferably alginate.

In a preferred embodiment the substance contains 0.5 to 60 wt. % ofpolymer. As a result of the formation of the material being carried outby solidification and consolidation, different concentrations ofpolymers can be used. The polymer concentrations can correspond to thenatural tissue which the material reproduces. For a high strength of thematerial, higher concentrations of polymers, above all of structuralproteins, such as collagens, are preferred. In a further preferredembodiment the substance therefore contains 0.8 to 10 wt. % of polymer,preferably approx. 3 wt. % of polymer.

In a preferred embodiment the first and/or a further substance containsat least one compound selected from the group consisting of ceramics,salts, metal oxides, semi-metal oxides, non-metal oxides, catalysts,proteins, growth factors, medicinal active compounds, lipids,surfactants, buffer substances and mixtures thereof. According to thefunctions and physical properties which the layers of the finishedmaterial are to be given, one or more substances can contain furthercompounds. Compounds having a medicinal and/or biological action canpreferably be integrated into medical materials. These include, forexample, antibiotics, anti-inflammatories, antimycotics, β-lactams, suchas penicillins, cephalosporins, monobactams and carbapenems,glycopeptides, such as vancomycins and teicoplanins, polyketides, such atetracyclines and macrolides, polypeptides, such as polymyxins,bacitracin and tyrothricin, quinolones, sulphonamides, aminoglycosides,streptomycins, amphenicols, aureomycins, non-steroidanti-inflammatories, glucocorticoids and polyene antimycotics, such asamphotericin B. After implantation or placing of the material in thebody, these compounds have a local action, which means that a systemictreatment, such as, for example, by enzyme inhibitors orimmunorepressants, can be prevented or reduced. If the material is usedfor implantation purposes, for example as a support matrix for chondraldefects, growth factors, such as TGF, BMP, GDF, FGF, IGF, annexin, MMP,PDGF, EGF, GMCSF, VEGF, HGF, interleukins, NGF and CSF, and/or compoundswhich promote the cell migration, are preferably integrated into thematerial. The migration of tissue cells into the material and theformation of extracellular matrix are thereby promoted. This can lead tointegration of the material into the tissue and also to a completere-establishment of the cartilage.

In a preferred embodiment the sublimable compound has a melting point of≦450° C., preferably ≦90° C., further preferred from −200° C. to +30°C., further preferred from −100° C. to +20° C. Preferably, thesublimable compound is liquid at room temperature, which allows a simpleprocessing and preparation of the substances.

In a preferred embodiment the sublimable compound is selected from thegroup consisting of aqueous solvents, polar solvents, non-polarsolvents, organic acids, organic bases, mineral acids and mineral bases.The sublimable compound can be added to the already dissolvedconstituents of the substance or, if the other constituents of thesubstance are present as a solid, these can be dissolved in thesublimable compound. In this case the sublimable compound is preferablya solvent in which the other constituents of the substance, for examplepolymers, are dissolved. Structural proteins in particular are onlysparingly soluble, so that they are preferably dissolved in a weak acid,for example in 0.25 to 5 M acetic acid, which also serves as thesublimable compound. The sublimable compound also influences thestructure of the pores in the finished material, because these areformed by the crystals of the sublimable compound. Since the crystalstructures of different sublimable compounds differ, the form of thepores, in particular their size and branching, can be influenced by thechoice of the sublimable compound. In a preferred embodiment thesublimable compound is water. Water is particularly suitable as thesublimable compound since it is universally available and alreadycrystallizes at comparatively high temperatures. Only a little coolingof the temperature-controllable bodies is therefore necessary in orderto solidify the substances which contain water as the sublimablecompound. As a result the entire process is advantageous in terms ofenergy and cost-effective.

In a further preferred embodiment the polar solvent is selected from thegroup consisting of ethanol, isopropanol, acetone, ether,dimethylsulphoxide, dimethylformamide, tetrahydrofuran,N-methyl-2-pyrrolidone, chloroform, 1,4-dioxane, acrylonitrile andacetonitrile.

In a further preferred embodiment the non-polar solvent is selected fromthe group consisting of benzene, toluene, methylene chloride, hexane,heptane and xylene.

In a further preferred embodiment the organic acid is selected from thegroup consisting of carboxylic acids, alkylcarboxylic acids, aceticacid, benzoic acid and alkylsulphonic acids.

In a further preferred embodiment the mineral acid is selected from thegroup consisting of sodium hydroxide solution, potassium hydroxide, limewater, phosphoric acid and hydrochloric acid.

In a preferred embodiment the first substance and at least one furthersubstance, preferably every further substance, have the same sublimablecompound. If two substances of adjacent layers contain the samesublimable compound, the crystals on the surface of the first, alreadysolidified layer combine with the still liquid molecules of thesublimable compound in the second substance when this is applied to thealready solidified layer. This promotes growing of the crystals whichprotrude beyond the first already rigid layer into the next layer.Moreover, the crystal structures in the layers become similar to oneanother if the same sublimable compound is used, as a result of whichall the layers have uniform pores after the sublimation.

In a preferred embodiment at least one of the temperature-controllableor insulating bodies or the container has a microstructuring. The term“microstructuring” describes a structuring on the surface of atemperature-controllable or insulating body or of a container in theform of projections and/or depressions at a distance of a fewmicrometers. This structuring initiates the formation of crystallizationnuclei, by means of which the points at which the crystallization orsolidification first starts can be controlled. The solidificationspreads out further from the crystallization nuclei, by means of whichthe spatial orientation of the crystals and therefore of the later porescan be controlled. By microstructurings in the form of concentriccircles, curves, waves or lines, a corresponding arrangement of thepores within the material can be achieved. However, the microstructuringcan also serve to configure the surface of the material, for example inorder to promote the adhesion of cells on the material. If themicrostructuring serves to control the crystallization, it is preferablyapplied to the temperature-controllable body or to the container inwhich the substances are introduced, each of which is arranged at thecoldest point of the temperature gradient.

In a preferred embodiment the sublimation is carried out under apressure of ≦6 mbar and at a temperature of ≦0° C., preferably under apressure of from 10 Oar to 1 mbar and at a temperature of from −80° C.to −20° C., further preferred under a pressure of from 50 Oar to 90 μbarand at a temperature of from −60° C. to −30° C. Due to the sublimation,the sublimable compound which has crystallized out in the solidifiedlayers is converted from its solid phase into the gas phase. The gasformed is sucked off, so that instead of the crystals of the sublimablecompound hollow bodies in the form of pores remain. The sublimationpressure and the sublimation temperature depend on the sublimablecompound used and can be obtained from temperature-phase diagrams. Thesublimation of water is carried out under below 6 mbar and at below 0°C. These sublimation parameters are also suitable for aqueous solutionsof acids or bases, for example for 0.25 to 5 M acetic acid.

In a preferred embodiment the process further comprises the step ofarranging a layer of functionalized polymer fibres on the support matrixas outermost layer. The layer of functionalized polymer fibres canfunction as a friction-reducing surface, or final membrane. The functionas a friction-reducing surface can be intensified by the use oflubricins. By a fibre alignment parallel to the material surface,shearing forces arising there can be processed better by the material.It can be generated by functionalized polymer fibres (e.g. collagen typeI, II, III, V, VI, IX, X, XI, XII, XIV, XVI, or linear, branched orstar-shaped polymers based on polyethylene glycol). The layer offunctionalized polymer fibres can be applied to the material by means ofelectrostatic spinning, before or after consolidation thereof(Grafahrend et al., 2010).

In a further aspect the invention relates to a multi-layered materialwhich can be produced by the process according to the invention. Thematerial produced by the process comprises several layers which differin their functionality, their composition and/or their physicalproperties. The various layers are combined to a monolith, that is tosay one piece or a one-piece form, so that a monolithic structure havinganisotropic pores is formed. In this manner the material combinesseveral different regions and can thus meet complex requirements such asarise above all in the medical field. For example, the layered structureof a natural bone-cartilage structure can be reproduced with thematerial (FIG. 2). The material is moreover distinguished by ananisotropic pore structure which penetrates all the layers and impartsto it specific physical and biological properties. Due to theanisotropic pore structure the stability of the material is improved,and by the pores protruding through all the layers continuously, that isto say crossing the boundaries of the layers, the cohesion of theindividual layers is promoted and a delamination is prevented. The poresfurthermore enable the penetration of substances as far as into theinside of the material. A population of the material with cells is alsopossible due to the pores, since the continuous pores enable a migrationof the cells as far as into the inside of the material. This is ofimportance above all for the use of the material as a bone/cartilagereplacement for articular defects, since the population of the materialwith chondrocytes after implantation contributes towards thereconstruction of the cartilage and towards the complete integration ofthe implant. The material is moreover also particularly suitable forcultivation of cells, since it provides a three-dimensional structurewhich, in contrast to conventional two-dimensional cultivation vessels,imitates the natural environment of the cells. An adequate exchange ofnutrients and toxins can likewise be effected due to the anisotropicpore structure.

In a preferred embodiment the pores have a columnar, lamellar and/ordendritic structure. The structure of the pores is determined by thenature of the sublimable compound, of the other constituents of thesubstances, and by the temperatures of the temperature-controllablebodies or the temperature gradient. A lamellar morphology here is to beunderstood as meaning a solidified phase of the sublimable additiveswhich is arranged in the form of lamellae. A columnar morphologycorresponds to a rod- or prism-shaped manifestation of the solidifiedphase of the sublimable additives. If ramifications or branches protrudeout of side faces of a columnar morphology, this is a dendriticallysolidified phase of the sublimable additives. In contrast to columnar,lamellar and dendritic crystals, equiaxial crystals have a sphericaldendritic structure. Columnar pore structures increase the stability ofthe material, so that this is more resistant to deformations. Lamellarpores, on the other hand, facilitate the population of the material withcells.

In a preferred embodiment the at least two layers have a differentcomposition. Materials which have layers or regions with differentcompositions can perform several functionalities. This is of importancein particular for medical materials which are integrated into naturaltissue. Most of the tissues in the human or animal body combinedifferent properties or perform different functions by having variousregions of different cellular or extracellular composition. Thisapplies, for example, to the extracellular matrix, which on the one handsupports and holds together the organs, but also controls cell adhesionand stores water. By a medical material combining several layers ofdifferent chemical compositions, it can reproduce the multifunctionalityof natural tissue, for example the natural extracellular matrix.

In a preferred embodiment the at least two layers independently of eachother have at least one polymer. Polymers are preferred constituents ofthe layers since they form stable structures and networks byintermolecular bonds. Preferably, the polymer is a native polymer.

In a preferred embodiment the pores have a diameter of from 20 μm to 380μm, preferably from 50 μm to 120 μm, further preferred of approx. 80 μm.In order to ensure an efficient population of the material by cells, thepores should have a diameter of at least 20 μm. For materials inparticular which are used as a (bone)-cartilage replacement, poreshaving a larger diameter, for example of from 60 μm to 100 μm, areadvantageous because they not only enable the migration of the cellsinto the inside of the material, they also leave space for the formationof extracellular matrix inside the material. Interestingly, an efficientpopulation of materials having relatively narrow pore chambers, forexample having diameters of from 20 μm to 50 μm, is also possible if thecomposition of the materials is particularly similar to the naturalextracellular matrix.

In a further aspect the invention relates to a medical material havingat least two different layers, in which pores extend anisotropicallythrough at least two layers of the material. The medical material can beproduced by the process according to the invention and can be present inthe preferred embodiments described above for a multi-layered material.

In a further aspect the invention relates to the use of a multi-layeredmaterial as a medical material in which pores extend anisotropicallythrough at least two layers of the material. The material can beproduced by the process according to the invention and can be present inthe preferred embodiments described above for a multi-layered material.

In a further aspect the invention relates to the use of a multi-layeredmaterial as a medical support matrix in which pores extendanisotropically through at least two layers of the material. Due to theanisotropic pore structure which extends beyond the boundaries of thelayers through the entire material, this is particularly suitable as amedical support matrix because a complete population of the material bycells is possible.

In a further aspect the invention relates to the use of a multi-layeredmaterial as a(n) (osteo)chondral support matrix in which pores extendanisotropically through at least two layers of the material. Nativechondral structures are distinguished by their multi-layered structure.By the material according to the invention combining several differentlayers, which moreover are traversed by anisotropic pores, the materialnot only corresponds to the natural (osteo)chondral matrix in structure,it can also reproduce the matrix with respect to the compositions of thelayers.

In a further aspect the invention relates to the use of a multi-layeredmaterial as a meniscus support matrix, characterized in that poresextend anisotropically through at least two layers of the material. Themulti-layered porous material can be produced in the form of a meniscusby the process according to the invention. The structure of a naturalmeniscus is distinguished by the different composition of the outer andthe inner region of the meniscus and by the arrangement of the collagenfibrils running parallel to the peripheral edge. This structure isreproduced by the different layers and the orientation of the pores inthe support matrix. Thus, curved or slanting structures are formed bycentral crystals being overgrown by adjacent crystals during thesolidification. Such a structure can be obtained, for example, by usingsubstances which have a relatively high viscosity and aretemperature-controlled beforehand. The temperature control of thesubstances should be close to the temperature level of the warmertemperature-controllable body, for example 0.5° C. to 5° C. above thetemperature level of the warmer temperature-controllable body. In thismanner, after sublimation curved or slanted pores at an angle of up to90° to the temperature gradient are obtained.

A further aspect the invention relates to the use of a multi-layeredmaterial as an intervertebral disc support matrix in which pores extendanisotropically through at least two layers of the material. Bothmaterials with layers arranged above one another and those with layersarranged side by side or concentrically can be produced by the processaccording to the invention. The latter are suitable above all for anintervertebral disc support matrix. In accordance with the naturalintervertebral disc, such a support matrix has an inner layer whichcorresponds to the nucleus pulposus, and layers arranged concentricallyaround this which correspond to the inner and the outer annulusfibrosus. By using substances having different compositions, theparticular layers can be adapted to the physical properties of thedifferent regions of an intervertebral disc. In order to reproduce theporosity of the natural intervertebral disc, the inner layer hasisotropic pores, whereas the layers of the inner and outer annulusfibrosus have anisotropic pores. This promotes the stability of thematrix and its integration into natural tissue.

In a further aspect the invention relates to a process for theproduction of a multi-layered (osteo)chondral support matrix, comprisingthe steps of providing a temperature gradient by twotemperature-controllable bodies arranged opposite one another; arrangingand solidifying in the temperature gradient a first substance whichcontains at least one polymer, at least one glycosaminoglycan and atleast one sublimable compound in order to form a middle chondral zone;arranging and solidifying in the temperature gradient a second substancewhich contains at least one polymer, at least one glycosaminoglycan andat least one sublimable compound in order to form a lower chondral zonedirectly adjacent to the middle chondral zone; optionally arranging andsolidifying in the temperature gradient a third substance which containsat least one polymer, at least one alkaline earth metal phosphate and atleast one sublimable compound in order to form a subchondral zonedirectly adjacent to the lower chondral zone; subliming the compound andconsolidating the layers.

Both multi-layered chondral support matrices (without a subchondralzone) and osteochondral support matrices (with a subchondral zone) canbe produced by the process. The support matrices are suitable inparticular for treatment of chondral or osteochondral defects, since thechemical composition of the individual layers correspond to the naturallayers of cartilage, that is to say the upper chondral zone, the lowerchondral zone and the subchondral zone (FIG. 2). Analogously to theconstituents of the natural chondral matrix, the substances forformation of the upper and lower chondral zone contain at least onepolymer and at least one glycosaminoglycan and the substance forformation of the subchondral zone contains at least one polymer and atleast one alkaline earth metal phosphate. The scaffold of theextracellular matrix of the native articular cartilage substantiallyconsists of a subchondral zone containing calcium phosphate (transitionzone to the bone), from which collagen fibres project (FIG. 2). Indeeper-lying chondral areas (d) these are orientated normally to thebone surface, in the middle areas (m) this converts into a slanted fibrearrangement, which in turn when close to the surface (s) runs parallelto this. Together with the collagen fibre structure, the manifestationof the chondral cells (chondrocytes) and therefore also that of theircell associates (chondrones) also changes. While the chondrocytes in thedeeper (d) and middle (m) chondral areas are round in shape and formcolumnar chondrones, in the upper chondral zone (s) they have a flatshape, and are combined in the form of horizontally running chondrones.

After the sublimation the monolithic matrix is consolidated, for exampleby being subjected to wet chemical crosslinking by means of activatedcarbodiimides, isocyanates, complexing ions, non-enzymatic glycation orglutaraldehyde. Preferably, the crosslinking is carried out by means ofN-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidewith buffering by 2-morpholinoethanesulphonic acid. In thiscell-compatible “zero-length” crosslinking covalent bonds are formedbetween the collagens, without further compounds being incorporated.Further preferred, the support matrix is pre-crosslinkeddehydrothermally before the wet chemical crosslinking, for example underpressures of between 1*10⁻⁶ mbar and 100 mbar and at temperatures ofbetween 50° C. and 200° C.

In a preferred embodiment the process further comprises the step ofarranging a layer of functionalized polymer fibres on the supportmatrix. The layer of functionalized polymer fibres forms afriction-reducing surface which corresponds to the articular inside ofnative cartilage. It can be generated by functionalized polymer fibres(e.g. collagen type I, II, III, V, VI, IX, X, XI, XII, XIV, XVI, orlinear, branched and star-shaped polymers based on polyethylene glycol),which form a final sliding layer (CS). The layer of functionalizedpolymer fibres can be applied to the material by means of electrostaticspinning, before or after consolidation thereof.

Due to the aligned solidification, the multi-layered chondral supportmatrix is traversed by anisotropic pores. As a result an efficient cellmigration into the inside of the matrix is ensured, which contributesdecisively towards complete integration of the support matrix into thedefective cartilage and the functionality thereof. A further improvementin the population by cells can be obtained by a matrix compression. Thisis to be understood as meaning a deformation of the support structurewhich is caused by external mechanical pressure and exerts a suctioneffect on the cells on the basis of the capillary forces which arise,and draws these into the inside of the matrix.

In a preferred embodiment the at least one polymer which is present inthe first, second and/or third substance, independently of each other,is a collagen selected from the group consisting of collagen type I, II,III, V, VI, IX, X, XI, XII, XIV, XVI, preferably collagen type I or typeII. Collagens are the natural structural proteins of cartilage and formthe predominant solids content of all the zones of natural cartilage.Collagens which are obtained from mammals and purified are suitable forthe preparation of the substances of the precursors of the individuallayers. Collagens which have been broken down by enzymatic cleavage andcomminuted purified cartilage fragments and collagen fibrils arespecifically suitable. Preferably, the substances comprise 0.5 to 60 wt.%, further preferred 0.8 to 10 wt. % of collagens.

In a preferred embodiment the at least one glycosaminoglycan which ispresent in the first and/or second substance, independently of eachother, is selected from the group consisting of chondroitin sulphate,aggrecan, keratan sulphate, hyaluronic acid, proteoglycan 4, cartilageoligomeric matrix protein (COMP), fibromodulin, procollagen II, decorin,anchorin, hyaluronate, biglycan, thrombospondin, fibronectin andchondrocalcin. Preferably, the dry matter content of the substancelayers has a content of glycosaminoglycans of from 1 to 35% a highercontent of glycosaminoglycans leading to an increased resistance tocompressions of the support matrix.

In a preferred embodiment the at least one alkaline earth metalphosphate contained in the third substance is a calcium phosphate or amagnesium phosphate, preferably selected from the group consisting ofbruschite, monetite, hydroxylapatite, α-tricalcium phosphate,β-tricalcium phosphate, whitlockite, struvite, newberite andfarringtonite. Higher contents of alkaline earth metal phosphatesincrease the resistance of the support matrix to compressions. Thealkaline earth metal phosphates can be present in the form of alkalineearth metal phosphate crystallites, alkaline earth metal phosphatesubstrates or in the form of composite materials. Alkaline earth metalphosphates can be obtained, for example, by a cement reaction, by 3Drapid prototyping of alkaline earth metal phosphate powders or startingsubstances thereof, or by aligned solidification with subsequentsintering of substances comprising alkaline earth metal phosphate.Alkaline earth metal phosphate composite materials can be produced,inter alia, by bioplotting of a mixture of polymers and alkaline earthmetal phosphate, or compounds which react to give alkaline earth metalphosphates.

In a preferred embodiment the first, second and/or third substancelayer, independently of each other, contains antibiotics and/or growthfactors, such as, for example, TGF, BMP, GDF, IGF, annexin and MMP. Bythe entire production process taking place exclusively at lowtemperatures, it is particularly suitable for integration of medicinalactive compounds, which are usually heat-sensitive, into the individuallayers of the support matrix. The active compounds can also be presentin encapsulated form within the substances.

In a preferred embodiment the sublimable compound is acetic acid. Aceticacid is particularly preferred as the sublimable compound since as aweak acid it impairs protein structures less severely than strong acids,and the residues thereof can readily be removed by sublimation and havea comparatively good cell tolerability. The higher the concentration ofcollagen to be dissolved, the higher the molarities of acetic acidemployed. In a particularly preferred embodiment the sublimable compoundtherefore contains 0.25-4 M acetic acid, preferably 0.5-3 M acetic acid,further preferred 0.5 M acetic acid.

In a preferred embodiment the first, second and/or third substancecontains 0.5-60 wt. % of polymer, preferably 0.8-10 wt. % of polymer,further preferred 1-5 wt. % of polymer. Higher polymer contents lead toa more stabile structure of the support matrix and are thereforepreferred in particular for use in joints which are subjected to load bythe body weight, for example knee joints.

In a preferred embodiment the temperature gradient for the production of(osteo)chondral support matrices is between 5 and 10 K/mm, preferably 8K/mm.

The support matrix is suitable in particular for treatment of chondraland osteochondral defects, both in the form of matrix-coupled autologouschondrocyte transplantation (MACT) and at the time of implantation ofcell-free matrix. The support matrix according to the invention canmoreover be employed for cell cultivation.

In a further aspect the invention relates to a process for theproduction of a multi-layered meniscus support matrix, comprising thesteps of providing a temperature gradient by twotemperature-controllable bodies arranged opposite one another; arrangingand solidifying in the temperature gradient a first substance whichcontains collagen I, at least one glycosaminoglycan and at least onesublimable compound in order to form an outer meniscus region; arrangingand solidifying in the temperature gradient a second substance whichcontains collagen I, collagen II, at least one glycosaminoglycan and atleast one sublimable compound in order to form an inner meniscus regiondirectly adjacent to the outer meniscus region; subliming the compoundand consolidating the support matrix.

The support matrix obtained by the process corresponds to the naturalmeniscus both in its composition and in its microstructure.Temperature-controllable and insulating bodies or a container whichcorrespond(s) to the negative form of a meniscus are used forsolidification of the substances (FIG. 5 b). The meniscus-like outermanifestation of the support matrix is thereby obtained (FIG. 5 c). Dueto the different layers of the support matrix, the outer and innermeniscus region is reproduced (FIG. 5 a). The orientation of the poresin the support matrix reproduces the arrangement of the collagen fibrilsof the natural meniscus running parallel to the peripheral edge.

After the sublimation the monolithic matrix is consolidated, for exampleby being subjected to wet chemical crosslinking by means of activatedcarbodiimides, isocyanates, complexing ions, non-enzymatic glycation orglutaraldehyde. Preferably, the crosslinking is carried out by means ofN-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidewith buffering by 2-morpholinoethanesulphonic acid. In thiscell-compatible “zero-length” crosslinking covalent bonds are formedbetween the collagens, without further substances being incorporated.Further preferred, the support matrix is pre-crosslinkeddehydrotherrnally before the wet chemical crosslinking, for exampleunder pressures of between 1*10⁻⁶ mbar and 100 mbar and at temperaturesof between 50° C. and 200° C.

In a preferred embodiment the process further comprises the step ofarranging a layer of functionalized polymer fibres on the supportmatrix. These polymer fibres correspond to a friction-reducing outerlayer of the natural meniscus, which forms a final sliding layer on thesurface. The layer of functionalized polymer fibres can be applied tothe material by means of electrostatic spinning, before or afterconsolidation thereof.

In a preferred embodiment the temperature gradient for the production ofmeniscus support matrices is between 3 and 8 K/mm, preferably 5 K/mm.

The support matrix is suitable for treatment of meniscus defects. Theprocess according to the invention enables to produce a meniscusreplacement according to the individual circumstances of the patient'sjoints. For this, the temperature-controllable and insulating bodies ora container which determines the form of the support matrix is producedaccording to three-dimensional reconstructions of the meniscus defect ofthe patient. This form is used in order to produce an accurately fittingsupport matrix.

In a further aspect the invention relates to a process for theproduction of a multi-layered intervertebral disc support matrix,comprising the steps of providing a temperature gradient by twotemperature-controllable bodies arranged opposite one another; arranginga first layer which forms a core and is formed from a first substancewhich contains at least one polymer, at least one glycosaminoglycan andat least one sublimable compound; arranging and solidifying in thetemperature gradient a second substance which contains at least onepolymer, at least one glycosaminoglycan and at least one sublimablecompound in order to form an inner layer directly adjacent to the core;arranging and solidifying in the temperature gradient a third substancewhich contains at least one polymer, at least one glycosaminoglycan andat least one sublimable compound in order to form an outer layerdirectly adjacent to the inner layer; subliming the compound andconsolidating the layers.

The natural intervertebral disc structure substantially consists of aninner core, the nucleus pulposus, which is enclosed by fibrous lamellaewhich form the annulus fibrosus (FIG. 7 a). The nucleus pulposus (NP) isstructurally and mechanically isotropic and contains aproteoglycan-containing network of collagen type II. The annulusfibrosus consists of a large number of lamellae which consist ofcollagen type I in the outer part (outer annulus fibrosus, oAF) andcollagen type II in the inner part (inner annulus fibrosus, iAF).Together with the inner intervertebral disc structure, the manifestationof the cells which occur in the intervertebral disc also changes. Whilethe notochordal cells in the nucleus pulposus are round in shape, thecells in the inner annulus fibrosus are chondrocytic. The cells in theouter annulus fibrosus are described as fibrochondrocytes. Analogouslyto native tissue, the support matrix has constituents of theextracellular chondral matrix, which are combined into a biomimetic,monolithic support matrix. A non-aligned network is to be found in thecore of the matrix and corresponds to the nucleus pulposus. This issurrounded by two layers of different composition, which have a lamellarstructure and correspond to the inner and outer annulus fibrosus.

After the sublimation the monolithic matrix is consolidated, for exampleby being subjected to wet chemical crosslinking by means of activatedcarbodiimides, isocyanates, complexing ions, non-enzymatic glycation orglutaraldehyde. Preferably, the crosslinking is carried out by means ofN-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidewith buffering by 2-morpholinoethanesulphonic acid. In thiscell-compatible “zero-length” crosslinking covalent bonds are formedbetween the collagens, without further substances being incorporated.Further preferred, the support matrix is pre-crosslinkeddehydrothermally before the wet chemical crosslinking, for example underpressures of between 1*10⁻⁶ mbar and 100 mbar and at temperatures ofbetween 50° C. and 200° C.

In a preferred embodiment the process further comprises the step ofarranging a layer of functionalized polymer fibres on the supportmatrix. These polymer fibres correspond to the outer membrane whichsurrounds the natural intervertebral disc. The layer of functionalizedpolymer fibres can be applied to the material by means of electrostaticspinning, before or after consolidation thereof.

In a preferred embodiment the at least one polymer which is present inthe first, second and/or third substance, independently of each other,is a collagen selected from the group consisting of collagen type I, II,III, V, VI, IX, X, XI, XII, XIV, XVI, cellulose, chitosan, polylacticacid (D and/or L) or polyglycollic acid, polycaprolactone andpolyethylene glycol, preferably collagen type I or type II. Collagensare the natural structural proteins of cartilage and form thepredominant solids content of all the zones of natural cartilage.Collagens which are obtained from mammals and purified are suitable forthe preparation of the substances of the precursors of the individuallayers. Collagens which have been broken down by enzymatic cleavage andcomminuted purified cartilage fragments and collagen fibrils arespecifically suitable. In a further preferred embodiment the firstsubstance contains collagen type II, the second substance collagen typeII and the third substance collagen type I.

In a preferred embodiment the at least one glycosaminoglycan which iscontained in the first, second and/or third substance, independently ofeach other, is selected from the group consisting of chondroitinsulphate, aggrecan, keratan sulphate, hyaluronic acid, proteoglycan 4,cartilage oligomeric matrix protein (COMP), fibromodulin, procollagenII, decorin, anchorin, hyaluronate, biglycan, thrombospondin,fibronectin and chondrocalcin. Preferably, the dry matter content of thesubstances for the production of intervertebral disc support matriceshas a content of glycosaminoglycans of from 10 to 55%, a higher contentof glycosaminoglycans leading to an increased resistance of the supportmatrix to compressions.

In a preferred embodiment the first, second and/or third substancelayer, independently of each other, contains antibiotics and/or growthfactors, for example, TGF, BMP, GDF, IGF, annexin and MMP. By the entireproduction process taking place exclusively at low temperatures, it isparticularly suitable for integration of medicinal active compounds,which are usually heat-sensitive, in the individual layers of thesupport matrix. The active compounds can also be present in encapsulatedform within the substances.

In a preferred embodiment the sublimable compound is acetic acid. Aceticacid is particularly preferred as the sublimable compound since as aweak acid it impairs protein structures less severely than strong acids,and the residues thereof can readily be removed by sublimation and havea comparatively good cell tolerability. The higher the concentration ofcollagens to be dissolved, the higher the molarities of acetic acidemployed. In a particularly preferred embodiment the sublimable compoundtherefore contains 0.25-4 M acetic acid, preferably 0.5-3 M acetic acid,further preferred 0.5 M acetic acid.

In a preferred embodiment the first, second and/or third substancecontains 0.5-60 wt. % of polymer, preferably 0.8-20 wt. % of polymer,further preferred 1-15 wt. % of polymer. Higher polymer contents lead toa more stable structure of the support matrix.

In a preferred embodiment the dry matter of the first, second and/orthird substance, independently of each other, contains 5%-65% ofglycosaminoglycans, such as, for example, chondroitin sulphate. In afurther preferred embodiment the dry matter of the first substance (NPprecursor) contains 10%-65% of glycosaminoglycans, the dry matter of thesecond substance (iAF precursor) contains 10%-55% of glycosaminoglycansand the dry matter of the third substance (oAF precursor) contains5%-30% of glycosaminoglycans.

In a preferred embodiment for the production of intervertebral discsupport matrices the temperature gradient is between 0.25 K/mm and 10K/mm.

In a preferred embodiment the interpolated solidification rate is from0.1×10⁻² mm/s to 10×10⁻² mm/s.

In a preferred embodiment the first layer is formed by wet chemicalcrosslinking. This is carried out, for example, by addition of acarbodiimide solution. The carbodiimide solution is present in the formof an ethanol/water mixture which contains 5-40 mM N-hydroxysuccinimide,50-250 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 100-350 mM2-morpholinoethanesulphonic acid. The pre-consolidated NP precursor isthen placed within a container which contains a further shaping,insulating body, and the free space between the pre-consolidated NPprecursor and the insulating body is filled with the second substance(iAF precursor).

In an alternative embodiment the first layer is formed by solidifyingthe first substance in a non-aligned form, for example at a uniformtemperature or a very small temperature gradient of <0.5 K/mm. For this,the substance for production of the NP zone (NP precursor) issolidified, for example, in a non-aligned form within a shapinginsulating body, which in turn can be within a container. Afterconclusion of the non-aligned solidification, the insulating body can beremoved from the container and replaced by a further shaping insulatingbody.

The space thereby formed is filled with the iAF precursor and theprecursors are solidified. The pre-consolidated NP precursor is therebysolidified in a non-aligned form, while the iAF precursor is solidifiedin an aligned form. After conclusion of the aligned solidification, theinsulating body can be removed from the container. After thesolidification of the second substance (iAF precursor), the insulatingbody is removed and the third substance (oAF precursor) is arranged inthe free space and solidified in an aligned form.

In a preferred embodiment the solidification of the precursors iscarried out within a microstructured container. The formation ofcrystallization nuclei of the sublimable additives preferentially arisesat points on the container base which are determined by themicrostructuring. Due to a corresponding microstructuring, the spatialorientation of the solidification forms of the sublimable additives iscontrolled. This leads, for example, to lamellar structures of theprecursors solidified in an aligned form (iAF and oAF), whichconcentrically enclose the precursor solidified in a non-aligned form(NP).

The support matrix is suitable for treatment of intervertebral discdefects, for example in the form of matrix-coupled cell transplantation.The support matrix according to the invention can moreover be employedfor cell cultivation.

EXAMPLES 1. Construction of the Solidification Apparatus

The solidification apparatus is constructed as shown in FIG. 1. Peltierelements coupled to heat exchangers generate a temperature gradientwhich is controlled by regulation of the flow of current in the Peltierelements. Successive precursors (e.g. subchondral (SC) suspension, deepchondral zone (CD) suspension and middle chondral zone (CM) suspension)are introduced into the sample chamber and thus placed within thetemperature gradient. Due to the temperature gradient a unidirectionalgrowth of ice crystals occurs within the precursors. The apparatus canfurther contain temperature-controllable bodies, as well as a containercontaining the precursors.

The sample chamber (FIG. 1 b) is in the centre of the insulation unit I1and is in thermal contact with the temperature-controllable bodies W1and W2, which are located both above and below the sample chamber. Thetemperature-controllable bodies W1 and W2 are within the insulation unitI2 and are coupled to the Peltier elements P1 and P2, which are fixed bythe insulation units I3 and I4. This inner assembly is in the centre ofthe heat exchanger ring A2, which together with the heat exchanger unitsA1 and A3 forms an outer assembly.

2. Osteochondral Support Matrix for Treatment of Articular ChondralDefects

2.1. Preparation of the Precursors

The precursors of the individual layers which reproduce the middlechondral zone, the deep chondral zone and the subchondral zone had thefollowing compositions:

middle chondral zone (CM):

-   -   1.0 wt. % of collagen type II    -   0.16 wt. % of chondroitin sulphate and    -   0.5 M acetic acid as the sublimable compound;        deep chondral zone (CD):    -   1.0 wt. % of collagen type II    -   0.2 wt. % of chondroitin sulphate and    -   0.5 M acetic acid as the sublimable compound;        subchondral zone (SC)    -   0.8 wt. % of collagen type I    -   0.8 wt. % of absorbable calcium phosphate phase bruschite and    -   0.5 M acetic acid as the sublimable compound.

Lyophilized collagen type II or collagen type I was used for thepreparation of the individual precursors. The constituents of theprecursors were stirred in acetic acid at room temperature for 30 minand then left to swell for 24 h at 5° C. Before use, the precursors weretemperature-controlled at 15° C. beforehand.

2.2 Freeze-Structuring of the Precursors

The freeze-structuring was carried out with a solidification apparatusas described in Example 1. A polystyrene cell culture dish was employedas a container for accommodating the precursors in the innerconstructional unit of the solidification apparatus. By electricalregulation of the Peltier elements, an external temperature gradient of8 K/mm with T_(Peltier1) −40° C. (lower Peltier element) andT_(Peltier2) 24° C. (upper Peltier element) was established. As soon asthe inner constructional unit and the container were close to thermalequilibrium, 2 ml of the precursor for the subchondral zone wereinjected into the container and solidified for 20 min. 2 ml of theprecursor of the deep chondral zone were then injected into thecontainer, so that the deep chondral zone was formed directly on thesubchondral zone. After a further 20 min 2 ml of the precursor of theupper chondral zone were injected into the container, i.e. directly onto the lower chondral zone. The precursors were solidified for a further20 min.

The container with the solidified precursors was then removed from thesolidification apparatus and if appropriate intermediately stored at−20° C.

2.3 Lyophilization of the Precursors Solidified in an Aligned Manner

The container with the solidified precursors was introduced into theworking volume of a running lyophilizer. The solidified precursors werelyophilized under a pressure of 0.08 mbar and at a temperature of −60°C. for 24 h. The solidified sublimable constituents of the precursorswere thereby sublimed out of these and removed.

2.4 Consolidation of the Material

The solidified precursors were pre-crosslinked by a dehydrothermalprocess under a pressure of 0.08 mbar and at a temperature of 105° C.for 210 min. The material was then consolidated further by wet chemicalcrosslinking. For this, the solidified and pre-crosslinked precursorswere placed in a pressure container and, after the working pressure of100 mbar was reached, 100 μl/mg of material of a carbodiimide solutionwere added. A 2:3 ethanol/water mixture which contained 21 mMN-hydroxysuccinimide, 52 mM1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 200 mM2-morpholinoethanesulphonic acid was used as the carbodiimide solution.45 seconds after infiltration of the material pores the pressurecontainer was ventilated. After a reaction time of 24 h the supportmatrix was washed three times in distilled water.

2.5 Application of Functionalized Polymer Fibres

Functionalized polymer fibres were applied as a friction-reducingsurface (CS) to the middle chondral zone of the support matrix. Thesewere applied to the material by means of electrostatic spinning, afterconsolidation thereof. The experimental procedure was carried out asdescribed in Grafahrend et al. (2010).

2.6 Properties of the Support Matrix

The finished four-layered support matrix reproduces native cartilage,and comprises a subchondral zone, a deep chondral zone, a middlechondral zone and a friction-reducing surface (FIGS. 2, 3 and 8). Lightmicroscopy and electron microscopy images of the support matrix showelongated anisotropic pores thereof both in the alginate model (Example6, FIG. 3) and in the collagen support matrix (FIG. 4). These structuresare formed by the aligned crystals of acetic acid which were formed bysolidification of the precursors and subsequent sublimation thereof. Thefibre structure of the friction-reducing surface runs perpendicular tothis pore structure. (FIG. 8)

3. Osteochondral Support Matrix for Treatment of Articular ChondralDefects

3.1 Preparation of the Precursors

The precursors of the individual layers which reproduce the middlechondral zone, the deep chondral zone and the subchondral zone had thefollowing compositions:

middle chondral zone (CM):

-   -   2.5 wt. % of collagen type II    -   0.5 wt. % of chondroitin sulphate and    -   0.5 M acetic acid as the sublimable compound;        deep chondral zone (CD):    -   2.5 wt. % of collagen type II    -   0.8 wt. % of chondroitin sulphate and    -   0.5 M acetic acid as the sublimable compound;        subchondral zone (SC)    -   1.0 wt. % of collagen type I    -   0.5 wt. % of absorbable calcium phosphate phase bruschite and    -   0.5 M acetic acid as the sublimable compound.

The individual precursors were prepared as in Example 2.

3.2 Freeze-Structuring of the Precursors

The freeze-structuring was carried out as described in Example 2 at anexternal temperature gradient of 6.25 K/mm with T_(Peltier1) −32° C.(lower Peltier element) and T_(Peltier2) 7.5° C. (upper Peltier element)and an interpolated solidification rate following from this of 0.27×10⁻²mm/s.

3.3 Lyophilization of the Precursors Solidified in an Aligned Manner

The lyophilisation was carried out as described in Example 2 under apressure of 0.08 mbar and at a temperature of −60° C. for 17 h.

3.4 Consolidation of the Material

The consolidation of the material was carried out as described inExample 2.

4. Support Matrix for Treatment of Meniscus Defects

4.1 Preparation of the Precursors

The precursors of the individual layers which reproduce the outer (OM)and the inner meniscus region (IM) (FIG. 5 a) had the followingcompositions:

outer meniscus region (OM):

-   -   1.5 wt. % of collagen type I    -   0.012 wt. % of chondroitin sulphate and    -   0.5 M acetic acid as the sublimable compound;        inner meniscus region (IM):    -   1.8 wt. % of collagen type II    -   1.2 wt. % of collagen type I    -   0.06 wt. % of chondroitin sulphate and    -   0.5 M acetic acid as the sublimable compound.

The individual precursors were prepared as in Example 2.

4.2 Freeze-Structuring of the Precursors

The freeze-structuring was carried out with a solidification apparatusas described in Example 1. A container which represented the negativeform of a meniscus (FIG. 5 b) and contained an insulating bodyfunctioning as a place-holder for the inner meniscus region was insertedinto the inner constructional unit of the solidification apparatus. Theprecursor of the outer meniscus region was first introduced into thiscontainer and solidified. The insulating body was then removed and theprecursor of the inner meniscus region was introduced and solidified.Due to the form of the container, the support structure formed acquiredthe form of a meniscus (FIG. 5 c).

By electrical regulation of the Peltier elements, an externaltemperature gradient of 4.5 K/mm with T_(Peltier1)=−20° C. (lowerPeltier element) and T_(Peltier2)=16° C. (upper Peltier element) wasestablished. An insulating body which served as a place-holder and hadthe form of the inner meniscus region was placed in the container. Assoon as the inner constructional unit and the container with theinsulating body were close to thermal equilibrium, 4 ml of the precursorfor the outer meniscus region were injected into the container andsolidified for 25 min. The inner edge of the outer meniscus region wasthereby formed. After solidification of the precursor of the outermeniscus region, the place-holder was removed from the container and 2ml of the precursor for the inner meniscus region were injected into thecontainer so that the inner meniscus region was formed directly on theouter meniscus region. The precursors were solidified for a further 15min.

The container with the solidified precursors was then removed from thesolidification apparatus and if appropriate intermediately stored at−20° C. until the further processing.

4.3 Lyophilization of the Precursors Solidified in an Aligned Manner

The lyophilisation was carried out as described in Example 2 under apressure of 0.08 mbar and at a temperature of −60° C. for 24 h.

4.4 Consolidation of the Material

The consolidation of the material was carried out as described inExample 2.

4.5 Properties of the Support Matrix

The finished two-layered support matrix corresponds to the outer form(FIG. 5 c) and the inner structure of a native meniscus (FIG. 6 a, b).The support matrix is traversed by lamellar pores (FIG. 6 a, b). Onsolidification of the precursors of the meniscus regions overgrowing ofthe central crystals by adjacent crystals occurs, as a result of whichlamellar pores corresponding to the native tissue structure runpredominantly horizontally through the meniscus support matrix.

5. Support Matrix for Treatment of Intervertebral Disc Defects

5.1 Preparation of the Precursors

The precursors of the individual layers which reproduce the nucleuspulposus (NP), the inner annulus fibrosus (iAF) and the outer annulusfibrosus (oAF) had the following compositions:

nucleus pulposus (NP):

-   -   4 wt. % of collagen type II    -   2 wt. % of chondroitin sulphate and    -   0.5 M acetic acid as the sublimable compound;        inner annulus fibrosus (iAF):    -   2 wt. % of collagen type II    -   0.8 wt. % of chondroitin sulphate and    -   0.5 M acetic acid as the sublimable compound;        outer annulus fibrosus (oAF):    -   1 wt. % of collagen type I    -   0.15 wt. % of chondroitin sulphate and    -   0.5 M acetic acid as the sublimable compound.

The individual precursors were prepared as in Example 2.

5.2 Freeze-Structuring of the Precursors

The freeze-structuring is carried out with a solidification apparatus asdescribed in Example 1. A container which corresponds in form to anintervertebral disc is arranged in the inner constructional unit of thesolidification apparatus. On the base of this container is amicrostructure consisting of linear depressions. These spread out in theradial direction, starting from the region of the nucleus pulposus (NP).The microstructure serves as a crystallization point for thesolidification of the precursors of the inner annulus fibrosus (iAF) andthe outer annulus fibrosus (oAF).

The microstructured container was laid in the inner constructional unitof the solidification apparatus, insulating bodies functioning asplace-holders for the iAF and oAF. The place-holders occupied approx.36% of the volume available for production of the support matrix. Byelectrical regulation of the Peltier elements, an external temperaturegradient of 0.25 K/mm was first established (T_(Peltier1)=−22° C.;T_(Peltier2)=−20° C.). As soon as the inner constructional unit togetherwith the shaping bodies contained therein was close to thermalequilibrium, 0.7 ml of the precursor of the NP was filled into thecentre of the container within the sample chamber and solidified for 15min. By electrical regulation of the Peltier elements, an externaltemperature gradient of 8.5 K/mm was then established (T_(Peltier1)=−40°C.; T_(Peltier2)=28° C.). As soon as the inner constructional unittogether with the shaping bodies contained therein was close to thermalequilibrium, the place-holder for the iAF precursor was removed and 1.2ml of the precursor of the iAF were filled in between the layercorresponding to the NP and the insulating place-holder for the oAFprecursor. The place-holder for the oAF precursor was located on theouter edge of the container and filled approx. 18% of the volume of thecontainer. After a further 10 min of freeze-structuring the place-holderwas removed and in its place 1.5 ml of the precursor of the oAF were fedin. After a further 10 min of freeze-structuring the container with theprecursors solidified in a common aligned manner was removed and ifappropriate intermediately stored at −20° C. until the furtherprocessing.

5.3 Lyophilization of the Precursors Solidified in an Aligned Manner

The lyophilisation was carried out as described in Example 2 under apressure of 0.08 mbar and at a temperature of −60° C. for 17 h.

5.4 Consolidation of the Material

The consolidation of the material was carried out as described inExample 2.

5.5 Properties of the Support Matrix

Because of the only very small temperature gradient during thesolidification of the support matrix region corresponding to the NP,non-aligned solidification occurs. As a result, this region has anisotropic pore structure. The regions of the iAF and the oAF which areadjacent to this, on the other hand, have a lamellar anisotropic porestructure due to the aligned solidification in the temperature gradient.Due to the microstructuring on the base of the container, theanisotropic pores show a pattern arranged concentrically around theregion of the NP (FIG. 7 b).

6. Alginate Model for a Support Matrix for Treatment of Meniscus Defects

6.1 Preparation of the Precursors

The precursors of the individual layers comprised, for model purposes,5.5% alginate, dissolved in distilled water.

6.2 Freeze-Structuring of the Precursors

The freeze-structuring was carried out with a solidification apparatusas described in Example 1. A container which represented the negativeform of a meniscus (FIG. 5 b) was inserted into the inner constructionalunit of the solidification apparatus. The precursor of the outermeniscus region was first introduced into this container and solidified,and the precursor of the inner meniscus region was then introduced andsolidified. Due to the form of the container, the support structureformed acquired the form of a meniscus (FIG. 5 c).

By electrical regulation of the Peltier elements, an externaltemperature gradient of 1.8 K/mm with T_(Peltier1)=−20° C. (lowerPeltier element) and T_(Peltier2)=5° C. (upper Peltier element) wasestablished. As soon as the inner constructional unit and the containerwith an insulating body which served as a place-holder and had the formof the inner meniscus region were close to thermal equilibrium, 8 ml ofthe precursor for the outer meniscus region were injected into thecontainer and solidified for 30 min. The inner edge of the outermeniscus region was thereby formed. After solidification of theprecursor of the outer meniscus region, the place-holder was removedfrom the container and 4 ml of the precursor for the inner meniscusregion were injected into the container so that the inner meniscusregion was formed directly on the outer meniscus region. The precursorswere solidified for a further 15 min.

The container with the solidified precursors was then removed from thesolidification apparatus and if appropriate intermediately stored at−20° C. until the further processing.

6.3 Lyophilization of the Precursors Solidified in an Aligned Manner

The lyophilisation was carried out as described in Example 2 under apressure of 0.08 mbar and at a temperature of −60° C. for 24 h.

6.4 Consolidation of the Material

The consolidation of the material was carried out by wet chemicalcrosslinking. For this, the freeze-dried structure was placed in apressure container and, after the working pressure of 100 mbar wasreached, 50 μl/mg of material of a 1 M CaCl₂ solution were added. 45seconds after infiltration of the material pores the pressure containerwas ventilated. After a reaction time of 24 h the support matrix waswashed three times in distilled water.

7. Alginate Model for a Support Matrix for Treatment of IntervertebralDisc Defects

7.1 Preparation of the Precursors

The precursors of the individual layers consisted of, for modelpurposes, 5.5% alginate, dissolved in distilled water, and had differentadded dyes for visual differentiation of the individual layers. Beforeuse, the precursors were temperature-controlled at 15° C. beforehand.

7.2 Freeze-Structuring of the Precursors

The freeze-structuring was carried out as described in Example 5.

7.3 Lyophilization of the Precursors Solidified in an Aligned Manner

The lyophilisation was carried out as described in Example 2 under apressure of 0.08 mbar and at a temperature of −60° C. for 24 h.

7.4 Consolidation of the Material

The consolidation of the material was carried out as described inExample 6.

REFERENCES

-   A. Tampieri, M. Sandri, E. Landi, D. Pressato, S. Francioli, R.    Quarto, et al., Biomaterials Design of graded biomimetic    osteochondral composite scaffolds, Biomaterials. 29 (2008)    3539-3546.-   T. J. Klein, S. C. Rizzi, J. C. Reichert, N. Georgi, J. Malda, W.    Schuurman, et al., Strategies for Zonal Cartilage Repair using    Hydrogels, Macromolecular Bioscience. 9 (2009) 1049-1058.-   D. Grafahrend, K.-H. Heffels, M. V. Beer, P. Gasteier, M. Möller, G.    Boehm, et al., Degradable polyester scaffolds with controlled    surface chemistry combining minimal protein adsorption with specific    bioactivation, Nature Materials. 10 (2010) 67-73.-   DE 197 51 031 A1-   EP 1 858 562 B1

The invention claimed is:
 1. A process for the production of amulti-layered material having anisotropic pores, the process comprisingthe steps of: (a) providing a temperature gradient between twotemperature-controllable bodies arranged opposite one another; (b)arranging in the temperature gradient a first substance containing atleast one sublimable compound and solidifying the first substance toform a first layer; (c) arranging in the temperature gradient a secondsubstance containing at least one sublimable compound adjacent to thefirst substance and solidifying the second substance to form a secondlayer adjacent to the first layer; (d) subliming the sublimablecompounds of the adjacent first and second layers to form a monolithicsupport matrix of the first and second layers having pores generated bythe subliming, the pores extending continuously and anisotropicallythroughout the first and second layers of the monolithic support matrix,and the pores penetrating the first and second layers of the supportmatrix; and (e) consolidating the support matrix, wherein the arrangingof the second substance is conducted over the first substance,side-by-side with the first substance, or concentrically around thefirst substance without the solidifying of the first and secondsubstances being interrupted.
 2. The process of claim 1, wherein thetemperature gradient is between 0.5 K/mm and 200 K/mm.
 3. The process ofclaim 2, wherein the temperature gradient is between 2.5 K/mm and 25K/mm.
 4. The process of claim 2, wherein the temperature gradient isbetween 5 K/mm and 15 K/mm.
 5. The process of claim 1, wherein the firstand/or the second substance, independently of each other, contains atleast one polymer or monomers thereof.
 6. The process of claim 5,wherein the polymer is selected from the group consisting of peptides,proteins, polysaccharides, and mixtures thereof.
 7. The process of claim6, wherein the polymer is a protein, and the protein is a structuralprotein.
 8. The process of claim 1, wherein the sublimable compound ofthe first and/or the second substance, independently of each other, isselected from the group consisting of aqueous solvents, polar solvents,non-polar solvents, organic acids, organic bases, mineral acids, andmineral bases.
 9. The process of claim 1, wherein the sublimablecompounds of the first substance and the second substance are the samesublimable compound.
 10. The process of claim 1, wherein at least one ofthe temperature-controllable bodies has a microstructuring.
 11. Theprocess of claim 1, further comprising the step of: arranging a layer offunctionalized polymer fibres on the support matrix as an outermostlayer.
 12. The process of claim 1, wherein the consolidating is carriedout by wet chemical crosslinking, dehydrothermal processes, enzymaticcrosslinking, non-enzymatic glycation, UV irradiation, gammairradiation, sintering, infiltration, or a combination thereof.
 13. Theprocess of claim 1, wherein as a result of the solidifying of the secondsubstance adjacent to the first substance, crystals of the sublimablecompounds grow uniformly successively throughout the first and secondlayers, and wherein the crystals are removed as a result of thesubliming so as to leave the continuous and anisotropic pore structurein the support matrix.
 14. The process of claim 1, wherein the supportmatrix is a chondral support matrix, an osteochondral support matrix, ameniscus support matrix, or an intervertebral disc support matrix. 15.The process of claim 1, wherein the first and second layers of thesupport matrix differ in composition, functionality, and physicalproperties.
 16. The process of claim 1, wherein step (c) furthercomprises arranging in the temperature gradient a third substancecontaining at least one sublimable compound adjacent to the secondsubstance and solidifying the third substance to form a third layeradjacent to the second layer, wherein step (d) further comprisessubliming the sublimable compound of the third layer to form themonolithic support matrix additionally of the third layer, the poresextending anisotropically throughout the first, second, and third layersof the monolithic support matrix, and the pores penetrating the first,second, and third layers of the support matrix, and wherein thearranging of the third substance is conducted without the solidifying ofthe first, second, and third substances being interrupted.
 17. Theprocess of claim 16, wherein the first, second, and third layers of thesupport matrix differ in composition, functionality, and physicalproperties.
 18. The process of claim 16, wherein the arranging of thethird substance is conducted over the second substance, side-by-sidewith the second substance, or concentrically around the secondsubstance.
 19. The process of claim 16, wherein the pores extendthroughout the first, second, and third layers of the support matrix.